PROJECT SUMMARY The heart's natural pacemaker, the sinoatrial node (SAN), generates a precisely timed electrical impulse that initiates each one of the roughly two billion normal heartbeats in a typical human life span. At present, there are no therapies that can delay, prevent, or reverse SAN failure, which remains the most common reason for permanent pacemaker implantation and a major contributor to the pathogenesis of atrial fibrillation. In part, this is because the basic mechanisms that maintain SAN function are still poorly understood. While it is well established that SAN output relies on the function of a small number of specialized pacemaker cells (PCs), little is known about how PCs acquire and maintain their unique functional properties. The primary reason for this knowledge gap is that PCs lack specific molecular markers, are few in number, and constitute only 1-2% of cells within the SAN, making them difficult to identify and isolate in sufficient quantities for molecular studies. The long-term goal of our lab is facilitate novel therapies for SAN failure and atrial fibrillation by developing a detailed understanding of the embryological origin of PCs, their unique gene regulatory networks, and the pathways that guide PCs to form and maintain a functional node. Our main hypothesis is that a combinatorial network of transcriptional activators required for PC development can be identified in-vivo and redeployed in the adult heart to reconstitute nodal function. To test this hypothesis, we have overcome the issue of low cell number by using ATAC-seq and CUT&RUN on isolated PCs. Second, our epigenetic data set led us to discover the first known discrete cis-regulatory element that is active specifically in pacemaker cells, permitting a more detailed characterization of the emergence of this lineage that was previously possible. By applying these tools to a series of novel mouse lines developed for this project, we will pursue two complementary lines of investigation. First, we will use our novel PC-specific regulatory element to trace the genetic and epigenetic signatures of PCs from their emergence in the embryo to a fully functional node. In an overlapping investigation, we will build on our preliminary findings that a remarkably small number of genetic loci differ in epigenetic state between PCs and working cardiomoycytes, raising the possibility that direct lineage conversion between working cardiomyocytes and PCs could be a viable regenerative strategy for SAN failure. To test this possibility, we have developed a gene delivery system in a reporter mouse line in which combinations of transcriptional regulators can be tested for their ability to activate PC genes in non-PCs and thereby effect functional lineage conversion and nodal regeneration. The significance of this project lies in its potential to develop an entirely new type of regenerative therapy for heart rhythm disorders. This project is innovative because it uses new technologies to overcome long-standing obstacles to understanding the origin and maintenance of heart rhythm, a long-standing and fundamental problem in animal biology.